Abstract:

Self-assembling peptide nanoparticles (SAPN) incorporating T-cell epitopes
and/or B-cell epitopes are described. The nanoparticles of the invention
consist of aggregates of a continuous peptidic chain comprising two
oligomerization domains connected by a linker segment wherein one or both
oligomerization domains incorporate T-cell epitopes and/or B-cell
epitopes within their peptide sequence. These nanoparticles are useful as
vaccines and adjuvants.

Claims:

1. A self-assembling peptide nanoparticle consisting of aggregates of a
multitude of building blocks of formula (I) consisting of a continuous
chain comprising a peptidic oligomerization domain D1, a linker segment
L, and a peptidic oligomerization domain D2D1-L-D2 (I)wherein D1 is a
peptide having a tendency to form oligomers (D1)m of m subunits D1,
D2 is a peptide having a tendency to form oligomers (D2)n of n
subunits D2, m and n each is a figure between 2 and 10, with the proviso
that m is not equal n and not a multiple of n, and n is not a multiple of
m, L is a bond or a short linker segment, either D1 or D2 or both D1 and
D2 is a coiled-coil oligomerization domain that incorporates one or more
T- and/or B-cell epitopes within the oligomerization domain, and wherein
D1, D2 and L are optionally further substituted.

2. The peptide nanoparticle according to claim 1 wherein the coiled-coil
oligomerization domain is consisting of heptad and/or undecad repeats.

3. The peptide nanoparticle according to claim 1 wherein the peptidic
oligomerization domain D1 at its N-terminal end and/or the peptidic
oligomerization domain D2 at its C-terminal end is substituted by one or
more additional B- and/or T-cell epitope, one or more other functional
peptide or protein, or one or more additional hapten or other functional
molecule.

4. The peptide nanoparticle according to claim 3 of the formulae
S1-D1-L-D2, D1-L-D2-S2, or S1-D1-L-D2-S2, wherein S1 and S2 are peptidic
substituents.

5. The peptide nanoparticle according to claim 3, consisting of identical
building blocks D1-L-D2, wherein at least one of the identical building
blocks carries one or more different substituent at the N-terminal end of
D1 and/or the C-terminal end of D2.

6. The peptide nanoparticle according to claim 1 wherein one of the
oligomerization domains D1 and D2 is the pentamerization domain of the
tryptophane zipper or a derivative thereof.

7. The peptide nanoparticle according to claim 1 wherein one of the
oligomerization domains D1 and D2 is the tetramerization domain of
tetrabrachion or a derivative thereof.

8. The peptide nanoparticle according to claim 1 wherein at least one of
the epitopes is a CTL epitope.

9. The peptide nanoparticle according to claim 1 wherein at least one of
the epitopes is a HTL epitope.

10. The peptide nanoparticle according claim 1 wherein at least one of the
epitopes is a B-cell epitope.

11. The peptide nanoparticle according to claim 1 wherein the sequence
D1-L-D2 comprises a series of optionally overlapping T- and/or B-cell
epitopes.

12. A composition comprising a peptide nanoparticle according to claim 1.

13. The composition of claim 12, wherein at least one of the B- or T-cell
epitopes is selected from the group consisting of:(a) an antigen suited
to induce an immune response against bacteria;(b) an antigen suited to
induce an immune response against viruses;(c) an antigen suited to induce
an immune response against parasites;(d) an antigen suited to induce an
immune response against cancer cells;(e) an antigen suited to induce an
immune response against allergens;(f) an antigen suited to induce an
immune response against addictions;(g) an antigen suited to induce an
immune response against diseases and metabolic disorders;(h) an antigen
suited to induce an immune response in a farm animals; and(i) an antigen
suited to induce an immune response in a pet.

16. The composition of claim 12, wherein at least one of the B- or T-cell
epitopes is selected from the influenza proteins hemagglutinin and/or M2.

17. The composition of claim 16, wherein the M2 B-cell epitope is the
tetrameric form of the extracellular portion of this protein M2e attached
to the N-terminal end of a tetrameric coiled-coil oligomerization domain
D1.

18. The composition of claim 17, wherein the tetrameric oligomerization
domain D1 is the tetrameric coiled-coil domain of tetrabrachion or a
derivative thereof.

19-20. (canceled)

21. The composition of claim 12, wherein at least one of the B-cell
epitopes is a sequence of between 8 and 48 residues that constitute a B
cell epitope of the Plasmodium falciparum circumsporozoite (CS) protein
said B cell epitope being comprised of two to about 12 repeats of the
amino acid residue sequence Asn-Ala-Asn-Pro or permutations thereof.

22-25. (canceled)

26. The composition of claim 12, wherein at least one of the B-cell
epitopes is a protein of HIV selected from the proteins gp41, gp120 or
gp160.

27. The composition of claim 12, wherein at least one of the B-cell
epitopes is a peptide of HIV selected from the binding sites of the
neutralizing antibodies 2F5 and/or 4E10 of gp41.

28. The composition of claim 12, wherein at least one of the B-cell
epitopes is a peptide of HIV selected from the V3-loop of gp120 of HIV.

37. The composition of claim 12, wherein at least one of the B-cell
epitopes is the Aβ-peptide or a fragment thereof comprising at least
the sequence of 6 amino acids starting with the N-terminal amino acids.

38. The composition of claim 12, wherein at least one of the B-cell
epitopes is angiotensin I or angiotensin II.

39. The composition of claim 12, wherein at least one of the B-cell
epitopes is grehlin.

40. The composition of claim 12, wherein at least one of the B-cell
epitopes is TNFα or a fragment thereof comprising at least the
sequence of 20 amino acids starting with the 4th N-terminal amino acid of
TNFα.

41. A method of vaccinating a human or non-human animal, which comprises
administering an effective amount of a peptide nanoparticle according to
claim 1 to a subject in need of such vaccination.

42. A monomeric building block of formula (I) consisting of a continuous
chain comprising a peptidic oligomerization domain D1, a linker segment
L, and a peptidic oligomerization domain D2D1-L-D2 (I)wherein D1 is a
peptide having a tendency to form oligomers (D1)m of m subunits D1,
D2 is a peptide having a tendency to form oligomers (D2)n of n
subunits D2, m and n each is a figure between 2 and 10, with the proviso
that m is not equal n and not a multiple of n, and n is not a multiple of
m, L is a bond or a short linker segment, either D1 or D2 or both D1 and
D2 is a coiled-coil oligomerization domain that incorporates one or more
T- and/or B-cell epitopes within the oligomerization domain, and wherein
D1, D2 and L are optionally further substituted.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to self-assembling peptide
nanoparticles incorporating B-cell epitopes and/or T-cell epitopes.
Furthermore, the invention relates to the use of such nanoparticles for
vaccination.

BACKGROUND OF THE INVENTION

[0002]The adaptive immune system has two different responses, the humoral
immune response and the cellular immune response. The first is
characterized by an antibody response in which these antibodies bind to
surface epitopes of pathogens while the latter is characterized by
cytotoxic T-lymphozytes (CTLs) that kill already infected cells. Both
immune responses are further stimulated by T-helper cells that activate
either the B-cells that are producing specific pathogen binding
antibodies or T-cells that are directed against infected cells.

[0003]The specificity of the interaction between the antibodies produced
by B-cells and the pathogen is determined by surface structures of the
pathogen, so called B-cell epitopes, while the specificity of the
interaction of CTLs with the infected target cell is by means of T-cell
epitopes presented on surface molecules of the target cell, the so-called
major histocompatibility complex class I molecules (MHC I). This type of
T-cell epitopes (CTL-epitopes) are fragments of the proteins from the
pathogen that are produced by the infected cell. Finally, the specificity
of the interaction of the T-helper cells with the respective B-cell or
CTL is determined by binding of receptor molecules of the T-helper cells
to the other type of T-cell epitopes (HTL-epitopes) presented by the MHC
class II molecules (MHC II) on the B-cells or CTL-cells.

[0004]Binding of the antibodies to the B-cell epitopes requires the B-cell
epitope to assume a particular three-dimensional structure, the same
structure that this B-cell epitope has in its native environment, i.e.
when it is on the surface of the pathogen. The B-cell epitope may be
composed of more than one peptide chain and is organized in a three
dimensional structure by the scaffold of the protein.

[0005]The T-cell epitopes, however, do not require a particular
three-dimensional structure, rather they are bound by the respective MHC
I or MHC II molecule in a very specific manner. CTL epitopes are trimmed
to a size of 9 amino acids in length for optimal presentation by the MHC
I molecules, while HTL epitopes make a similar interaction with the MHC
II molecules but may be longer than just 9 amino acids. Important in the
context of this invention is, that the binding of the epitopes to the MHC
molecules follows very particular rules, i.e. only peptides with specific
features will be able to bind to the respective MHC molecule and hence be
useful as epitopes. These features have been thoroughly investigated and
from the wealth of epitopes known, prediction programs have been
developed that are able to predict with high accuracy epitopes that are
able to bind to the MHC molecules. Peptide strings composed of several
such T-cell epitopes in a linear peptide chain are now being engineered
as vaccine candidates.

[0006]In general an efficient vaccine should induce a strong humoral as
well as a strong cellular immune response. It has been shown that by
repetitive antigen display of B-cell epitopes a strong humoral immune
response can be achieved. Virus-like particles (VLPs) can be used as an
efficient tool to present B-cell epitopes in a regular, repetitive and
rigid manner, and hence VLPs are now widely used for vaccine design.
Another approach for repetitive antigen display has been described in
Patent EP 1 594 469 B1. In this patent self-assembling peptide
nanoparticles (SAPN) composed of trimeric and pentameric protein
oligomerization domains have been engineered that repetitively display
B-cell epitopes on their surface. The B-cell epitopes were attached at
the end of the oligomerization domains in order to guarantee that the
B-cell epitopes are presented at the surface of the nanoparticles in
multiple copies. One of the most frequently encountered protein
oligomerization motif is the coiled-coil structural motif and this motif
can efficiently be used in the design of these SAPN.

SUMMARY OF THE INVENTION

[0007]The invention relates to self-assembling peptide nanoparticles
(SAPN) incorporating T-cell epitopes and/or B-cell epitopes. In
particular, nanoparticles of the invention consist of aggregates of a
continuous peptidic chain comprising two oligomerization domains
connected by a linker segment wherein one or both oligomerization domains
is a coiled-coil that incorporates T-cell epitopes and/or B-cell epitopes
within its peptide sequence. The invention further relates to a method of
vaccinating humans or non-human animals using such self-assembling
peptide nanoparticles incorporating T-cell epitopes and/or B-cell
epitopes.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1: The structure of mouse MHC II molecule I-Ad covalently
linked to an ovalbumin peptide (OVA323-339), which is a HTL epitope for
I-Ad. The MHC II protein is shown from the top in a C-alpha trace in
gray. The two helices forming the walls of the epitope binding sites are
flanking the bound peptide. The peptide is shown in an all-atom
ball-and-stick model in black. The peptide HTL epitope in its bound form
is in extended conformation as can be seen more clearly by the structure
of the peptide alone at the bottom of the figure.

[0009]FIG. 2: Schematic drawing of "even units" for trimeric and
pentameric oligomerization domains [left side, A)] and trimeric and
tetrameric oligomerization domains [right side, B)], respectively. The
number of monomers (building blocks) is defined by the least common
multiple (LCM) of the oligomerization states of the two oligomerization
domains D1 and D2 of the building blocks. In the even units the linker
segments of all building blocks will be arranged as closely to each other
as possible, i.e. as close to the center of the peptidic nanoparticle as
possible and hence the even units will self-assemble to a spherical
nanoparticle.

[0010]FIG. 3: Internal symmetry elements of the dodecahedron/icosahedron.
The rotational symmetry axes (2-fold, 3-fold and 5-fold) are displayed as
lines marked 2, 3 and 5. In A) a monomeric building block composed of
oligomerization domain D1 (left, coiled-coil domain with three-fold
symmetry), a linker segment L (bottom), and oligomerization domain D2
(right; coiled-coil domain with five-fold symmetry) is displayed such
that the internal symmetry elements of the oligomerization domains D1 and
D2 are superimposed onto the symmetry elements of the polyhedron. In B),
the complete coiled-coil domains D1 and D2 are displayed. The additional
symmetry objects generated by the 3-fold and the 5-fold rotational
symmetry elements of the polyhedron are displayed as cylinders while the
original molecule is displayed as a helix as in A).

[0011]FIG. 4: Dynamic lights scattering (DLS, A) and transmission electron
microscopy (TEM, B) of the self-assembled peptide nanoparticles formed
from peptides with the sequence SEQ ID NO:8, Example 1. The DLS analysis
shows size distribution with an average particle diameter of 32.01 nm and
polydispersity index of 12.9% (A). The TEM pictures (B) show
nanoparticles of the same size as determined by DLS.

[0012]FIG. 5: Transmission electron microscopy (TEM) of the self-assembled
peptide nanoparticles formed from peptides with the sequence SEQ ID
NO:10, Example 2. The TEM picture shows nanoparticles of the same size of
25 nm.

[0013]FIG. 6: Transmission electron microscopy (TEM) of the self-assembled
peptide nanoparticles formed from peptides with the sequence SEQ ID
NO:12, Example 3. The TEM picture shows nanoparticles of the size of
about 20 to 30 nm.

[0014]FIG. 7: Transmission electron microscopy (TEM) of the self-assembled
peptide nanoparticles formed from peptides with the sequences SEQ ID
NO:37 (panel A) and SEQ ID NO:38 (panel B), for a human and a chicken
influenza vaccine, respectively (Example 9). The TEM pictures show
nanoparticles of the size of about 25 nm.

[0015]FIG. 8: Transmission electron microscopy (TEM) of the self-assembled
peptide nanoparticles formed from peptides with the sequence SEQ ID
NO:41, Example 11. The TEM picture shows nanoparticles of the size of
about 25 nm.

DETAILED DESCRIPTION OF THE INVENTION

Monomeric Building Blocks

[0016]Self-assembling peptide nanoparticles (SAPN) are formed from a
multitude of monomeric building blocks of formula (I) consisting of a
continuous chain comprising a peptidic oligomerization domain D1, a
linker segment L and a peptidic oligomerization domain D2

D1-L-D2 (I)

wherein D1 is a synthetic or natural peptide having a tendency to form
oligomers (D1)m of m subunits D1, D2 is a synthetic or natural
peptide having a tendency to form oligomers (D2)n of n subunits D2,
m and n each is a figure between 2 and 10, with the proviso that m is not
equal n and not a multiple of n, and n is not a multiple of m, L is a
bond or a short linker chain selected from optionally substituted carbon
atoms, optionally substituted nitrogen atoms, oxygen atoms, sulfur atoms,
and combinations thereof; either D1 or D2 or both D1 and D2 is a
coiled-coil that incorporates one or more T-cell epitopes and/or a B-cell
epitope within the oligomerization domain, and wherein D1, D2 and L are
optionally further substituted.

[0017]A peptide (or polypeptide) is a chain or sequence of amino acids
covalently linked by amide bonds. The peptide may be natural, modified
natural, partially synthetic or fully synthetic. Modified natural,
partially synthetic or fully synthetic is understood as meaning not
occurring in nature. The term amino acid embraces both naturally
occurring amino acids selected from the 20 essential natural
α-L-amino acids, synthetic amino acids, such as α-D-amino
acids, 6-aminohexanoic acid, norleucine, homocysteine, or the like, as
well as naturally occurring amino acids which have been modified in some
way to alter certain properties such as charge, such as phosphoserine or
phosphotyrosine, or the like. In derivatives of amino acids the amino
group forming the amide bond is alkylated, or a side chain amino, hydroxy
or thio functions is alkylated or acylated, or a side chain carboxy
function is amidated or esterified.

[0019]m and n each is a figure between 2 and 10, with the proviso that m
is not equal n and not a multiple of n, and n is not a multiple of m.
Preferred combinations of n and m are combinations wherein m is 2 and n
is 5, or m is 3 and n is 4 or 5, or m is 4 and n is 5. Likewise preferred
combinations of n and m are combinations wherein m is 5 and n is 2, or m
is 4 or 5 and n is 3, or m is 5 and n is 4. Most preferred are
combinations wherein m or n is 5.

[0020]A coiled-coil is a peptide sequence with a contiguous pattern of
mainly hydrophobic residues spaced 3 and 4 residues apart, which
assembles to form a multimeric bundle of helices, as will explained in
more detail hereinbelow.

[0021]"A coiled-coil that incorporates T-cell and/or B-cell epitopes"
means that the corresponding epitope is comprised within an
oligomerization domain such that the amino acid sequences at the
N-terminal and the C-terminal ends of the epitope force the epitope to
adapt a conformation which is still a coiled-coil in line with the
oligomerization properties of the oligomerization domain comprising the
epitope. In particular, "incorporated" excludes a case wherein the
epitope is attached at either end of the coiled-coil oligomerization
domain.

[0022]In the context of this document the term T-cell epitopes shall be
used to refer to both CTL and HTL epitopes.

[0023]T-cell epitopes bind to the MHC molecules in extended conformation
(FIG. 1). Therefore, incorporating T-cell epitopes into an
α-helical coiled-coil (compare FIG. 3A and FIG. 1) is not a trivial
task of protein engineering. In this invention it is demonstrated how
these peptide sequences that have an extended conformation when bound to
the respective MHC molecule can nevertheless be incorporated into an
α-helical coiled-coil oligomerization domain.

[0024]Optional substituents of D1, D2 and L are e.g. B-cell epitopes,
targeting entities, or substituents reinforcing the adjuvant properties
of the nanoparticle, such as an immunostimulatory nucleic acid,
preferably an oligodeoxynucleotide containing deoxyinosine, an
oligodeoxynucleotide containing deoxyuridine, an oligodeoxynucleotide
containing a CG motif, or an inosine and cytidine containing nucleic acid
molecule. Other substituents reinforcing the adjuvant properties of the
nanoparticle are antimicrobial peptides, such as cationic peptides, which
are a class of immunostimulatory, positively charged molecules that are
able to facilitate and/or improve adaptive immune responses. An example
of such a peptide with immunopotentiating properties is the positively
charged artificial antimicrobial peptide KLKLLLLLKLK (SEQ ID NO: 63)
which induces potent protein-specific type-2 driven adaptive immunity
after prime-boost immunizations. A particular targeting entity considered
as substituent is an ER-targeting signal, i.e. a signal peptide that
induces the transport of a protein or peptide to the endoplasmic
reticulum (ER). Other optional substituents are, for example, an acyl
group, e.g. acetyl, bound to a free amino group, in particular to the
N-terminal amino acid, or amino bound to the free carboxy group of the
C-terminal amino acid to give a carboxamide function.

[0025]Optional substituents, e.g. those optional substituents described
hereinabove, are preferably connected to suitable amino acids close to
the free end of the oligomerization domain D1 and/or D2. On self-assembly
of the peptide nanoparticle, such substituents will then be presented at
the surface of the SAPN.

[0026]In a most preferred embodiment the substituent is another peptide
sequence S1 and/or S2 representing a simple extension of the peptide
chain D1-L-D2 at either end or at both ends to generate a combined single
peptide sequence of any of the forms S1-D1-L-D2, D1-L-D2-S2, or
S1-D1-L-D2-S2, wherein S1 and S2 are peptidic substituents as defined
hereinbefore and hereinafter. The substituents S1 and/or S2 are said to
extend the core sequence D1-L-D2 of the SAPN. Any such peptide sequence
S1-D1-L-D2, D1-L-D2-S2, or S1-D1-L-D2-S2 may be expressed in a
recombinant protein expression system as one single molecule.

[0027]A preferred substituent S1 and/or S2 is a B-cell epitope. Other
B-cell epitopes considered are hapten molecules such as a carbohydrate or
nicotine, which are likewise attached to the end of the oligomerization
domains D1 and/or D2, and hence will be displayed at the surface of the
SAPN.

[0028]Obviously it is also possible to attach more than one substituent to
the oligomerization domains D1 and/or D2. For example, considering the
peptide sequence S1-D1-L-D2-S2, another substituent may be covalently
attached to it, preferably at a location distant from the linker segment
L, either close to the ends of D1 and/or D2, or anywhere in the
substituents S1 and/or S2.

[0029]It is also possible to attach a substituent to the linker segment L.
In such case, upon refolding of the SAPN, the substituent will be located
in the inner cavity of the SAPN.

[0030]A tendency to form oligomers means that such peptides can form
oligomers depending on the conditions, e.g. under denaturing conditions
they are monomers, while under physiological conditions they may form,
for example, trimers. Under predefined conditions they adopt one single
oligomerization state, which is needed for nanoparticle formation.
However, their oligomerization state may be changed upon changing
conditions, e.g. from dimers to trimers upon increasing salt
concentration (Burkhard P. et al., Protein Science 2000, 9:2294-2301) or
from pentamers to monomers upon decreasing pH.

[0031]A building block architecture according to formula (I) is clearly
distinct from viral capsid proteins. Viral capsids are composed of either
one single protein, which forms oligomers of 60 or a multiple thereof, as
e.g. the hepatitis virus B particles (EP 1 262 555, EP 0 201 416), or of
more than one protein, which co-assemble to form the viral capsid
structure, which can adopt also other geometries apart from icosahedra,
depending on the type of virus (Fender P. et al., Nature Biotechnology
1997, 15:52-56). Self-assembling peptide nanoparticles (SAPN) of the
present invention are also clearly distinct from virus-like particles, as
they (a) are constructed from other than viral capsid proteins and (b)
that the cavity in the middle of the nanoparticle is too small to
accommodate the DNA/RNA of a whole viral genome.

[0033]One or both oligomerization domains D1 and D2, independently of each
other, are coiled-coil domains. A coiled-coil is a peptide sequence with
a contiguous pattern of mainly hydrophobic residues spaced 3 and 4
residues apart, usually in a sequence of seven amino acids (heptad
repeat) or eleven amino acids (undecad repeat), which assembles (folds)
to form a multimeric bundle of helices. Coiled-coils with sequences
including some irregular distribution of the 3 and 4 residues spacing are
also contemplated. Hydrophobic residues are in particular the hydrophobic
amino acids Val, Ile, Leu, Met, Tyr, Phe and Trp. Mainly hydrophobic
means that at least 50% of the residues must be selected from the
mentioned hydrophobic amino acids.

[0034]For example, in a preferred monomeric building block of formula (I),
D1 and/or D2 is a peptide of any of the formulae

[aa(a)-aa(b)-aa(c)-aa(d)-aa(e)-aa(f)-aa(g)]x (IIa)

[aa(b)-aa(c)-aa(d)-aa(e)-aa(f)-aa(g)-aa(a)]x (IIb)

[aa(c)-aa(d)-aa(e)-aa(f)-aa(g)-aa(a)-aa(b)]x (IIc)

[aa(d)-aa(e)-aa(f)-aa(g)-aa(a)-aa(b)-aa(d)]x (IId)

[aa(e)-aa(f)-aa(g)-aa(a)-aa(b)-aa(c)-aa(d)]x (IIe)

[aa(f)-aa(g)-aa(a)-aa(b)-aa(c)-aa(d)-aa(e)]x (IIf)

[aa(g)-aa(a)-aa(b)-aa(c)-aa(d)-aa(e)-aa(f)]x (IIg)

wherein aa means an amino acid or a derivative thereof, aa(a), aa(b),
aa(c), aa(d), aa(e), aa(f), and aa(g) are the same or different amino
acids or derivatives thereof, preferably aa(a) and aa(d) are the same or
different hydrophobic amino acids or derivatives thereof; and X is a
figure between 2 and 20, preferably 3, 4, 5 or 6.

[0036]A heptad is a heptapeptide of the formula
aa(a)-aa(b)-aa(c)-aa(d)-aa(e)-aa(f)-aa(g) (IIa) or any of its
permutations of formulae (IIb) to (IIg).

[0037]Preferred are monomeric building blocks of formula (I) wherein one
or both peptidic oligomerization domains D1 or D2 are

(1) a peptide of any of the formulae (IIa) to (IIg) wherein X is 3, and
aa(a) and aa(d) are selected from the 20 natural α-L-amino acids
such that the sum of scores from Table 1 for these 6 amino acids is at
least 14, and such peptides comprising up to 17 further heptads; or(2) a
peptide of any of the formulae (IIa) to (IIg) wherein X is 3, and aa(a)
and aa(d) are selected from the 20 natural α-L-amino acids such
that the sum of scores from Table 1 for these 6 amino acids is at least
12, with the proviso that one amino acid aa(a) is a charged amino acid
able to form an inter-helical salt bridge to an amino acid aa(d) or aa(g)
of a neighboring heptad, or that one amino acid aa(d) is a charged amino
acid able to form an inter-helical salt bridge to an amino acid aa(a) or
aa(e) of a neighboring heptad, and such peptides comprising up to two
further heptads. A charged amino acid able to form an inter-helical salt
bridge to an amino acid of a neighboring heptad is, for example, Asp or
Glu if the other amino acid is Lys, Arg or His, or vice versa.

[0038]Also preferred are monomeric building blocks of formula (I) wherein
one or both peptidic oligomerization domains D1 or D2 are selected from
the following preferred peptides:

(11) Peptide of any of the formulae (IIa) to (IIg) wherein aa(a) is
selected from Val, Ile, Leu and Met, and a derivative thereof, and aa(d)
is selected from Leu, Met and Ile, and a derivative thereof.(12) Peptide
of any of the formulae (IIa) to (IIg) wherein one aa(a) is Asn and the
other aa(a) are selected from Asn, Ile and Leu, and aa(d) is Leu. Such a
peptide is usually a dimerization domain (m or n=2).(13) Peptide of any
of the formulae (IIa) to (IIg) wherein aa(a) and aa(d) are both Leu or
both Ile. Such a peptide is usually a trimerization domain (m or
n=3).(14) Peptide of any of the formulae (IIa) to (IIg) wherein aa(a) and
aa(d) are both Trp. Such a peptide is usually a pentamerization domain (m
or n=5).(15) Peptide of any of the formulae (IIa) to (IIg) wherein aa(a)
and aa(d) are both Phe. Such a peptide is usually a pentamerization or
tetramerization domain (m or n=4 or 5).(16) Peptide of any of the
formulae (IIa) to (IIg) wherein aa(a) and aa(d) are both either Trp or
Phe. Such a peptide is usually a pentamerization domain (m or n=5).(17)
Peptide of any of the formulae (IIa) to (IIg) wherein aa(a) is either Leu
or Ile, and one aa(d) is Gln and the other aa(d) are selected from Gln,
Leu and Met. Such a peptide has the potential to be a pentamerization
domain (m or n=5).

(21) at least one aa(g) is selected from Asp and Glu and aa(e) in a
following heptad is Lys, Arg or His; and/or(22) at least one aa(g) is
selected from Lys, Arg and His, and aa(e) in a following heptad is Asp or
Glu, and/or(23) at least one aa(a to g) is selected from Lys, Arg and
His, and an aa(a to g) 3 or 4 amino acids apart in the sequence is Asp or
Glu. Such pairs of amino acids aa(a to g) are, for example aa(b) and
aa(e) or aa(f).

[0040]Coiled-coil prediction programs such as COILS
(http://www.ch.embnetorg/software/COILS_form.html; Gruber M. et al., J.
Struct. Biol. 2006, 155(2):140-5) or MULTICOIL
(http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi) can
predict coiled-coil forming peptide sequences. Therefore, in a preferred
monomeric building block of formula (I), D1 and/or D2 is a peptide that
contains at least a sequence two heptad-repeats long that is predicted by
the coiled-coil prediction program COILS to form a coiled-coil with
higher probability than 0.9 for all its amino acids with at least one of
the window sizes of 14, 21, or 28.

[0041]In a more preferred monomeric building block of formula (I), D1
and/or D2 is a peptide that contains at least one sequence three
heptad-repeats long that is predicted by the coiled-coil prediction
program COILS to form a coiled-coil with higher probability than 0.9 for
all its amino acids with at least one of the window sizes of 14, 21, or
28.

[0042]In another more preferred monomeric building block of formula (I),
D1 and/or D2 is a peptide that contains at least two separate sequences
two heptad-repeats long that are predicted by the coiled-coil prediction
program COILS to form a coiled-coil with higher probability than 0.9 for
all its amino acids with at least one of the window sizes of 14, 21, or
28.

[0043]In another preferred embodiment, one oligomerization domain D1 or D2
is the pentamerization domain (m or n=5) of COMP (Malashkevich V. N. et
al., Science 1996, 274:761-765) or a derivative thereof. This
pentamerization domain has the sequence
LAPQMLRELQETNAALQDVRELLRQQVKQITFLKNTVMECDACG (SEQ ID NO:1). Small
modifications of this domain are also envisaged. Such modifications may
be e.g. the substitution of amino acids at the outside of the pentamer at
positions aa(b), aa(c) or aa(f), preferably in position aa(f), by Cys for
the purpose of the formation of a disulfide bridge between adjacent
domains. Other modifications of surface amino acids of this domain may
include substitutions of amino acids for optimizing the interactions at
the interface between adjacent oligomerization domains such as
hydrophobic, hydrophilic or ionic interactions or covalent bonds like
disulfide bridges. Also shorter constructs of this domain, e.g. lacking
the C-terminal CDACG motif in which the cysteins form intermolecular
disulfide bridges at the C-terminus of this pentamerization domain are
also envisaged. Modification of amino acids affecting the oligomerization
state of this domain are also envisaged, resulting e.g. in a transition
from pentamer to tetramer. Yet other modifications of surface amino acids
of this domain may include substitutions of amino acids (e.g. by cysteine
or lysine) for the generation of attachment sites for functional groups.

[0044]In another preferred embodiment, one oligomerization domain D1 or D2
is the pentamerization domain (m or n=5) of the tryptophane zipper (Liu J
et al., Proc Natl Acad Sci USA 2004; 101(46):16156-61) or a derivative
thereof. This pentamerization domain has the sequence
SSNAKWDQWSSDWQTWNAKWDQWSNDWNAWRSDWQAWKDD WARWNQRWDNWAT (SEQ ID NO:2).
Small modifications of this domain are also envisaged. Such modifications
may be, e.g., the substitution of amino acids at the outside of the
pentamer at positions aa(b), aa(c) or aa(f), preferably in position
aa(f), by Cys for the purpose of the formation of a disulfide bridge
between adjacent domains. Other modifications of surface amino acids of
this domain may include substitutions of amino acids for optimizing the
interactions at the interface between adjacent oligomerization domains
such as hydrophobic, hydrophilic or ionic interactions, or covalent bonds
such as disulfide bridges. Also shorter constructs of this domain are
envisaged. Modification of amino acids affecting the oligomerization
state of this domain are also envisaged, resulting, for example, in a
transition from pentamerization domain to tetramerization domain
exchanging core residues Trp by Phe. Other core residue mutations as in
Example 10 are also considered, but at least 70% of the core positions
aa(a) and aa(d) have to be either a Trp or another aromatic amino acid.
Yet other modifications of surface amino acids of this domain may include
substitutions of amino acids (e.g. by cysteine or lysine) for the
generation of attachment sites for functional groups.

[0045]In another preferred embodiment, one oligomerization domain D1 or D2
is the tetramerization domain (m or n=4) of the coiled-coil domain of
tetrabrachion (Stetefeld J. et al., Nature Structural Biology, 2000;
7(9):772-776) or a derivative thereof. This tetramerization domain has
the sequence IINETADDIVYRLTVIIDDRYESLKNLITLRADRL MIINDNVSTILASG (SEQ ID
NO:64). The sequences of coiled coils are characterized by a heptad
repeat of seven residues with a 3,4-hydrophobic repeat. The next
periodicity that allows residues to assume quasi-equivalent positions
after a small number of turns is three turns or 11 residues. Based on the
presence of 11-residue repeats, the C-terminus of the surface layer
glycoprotein tetrabrachion from the hyperthermophilic archae-bacterium
Staphylothermus marinus forms a right-handed coiled coil structure. It
forms a tetrameric α-helical coiled coil stalk 70 nm long that is
anchored to the cell membrane at its C-terminal end. This tetrameric
coiled-coil contains a series of HTL epitopes (Example 9) and hence is
ideally suited as core oligomer of the self-assembling peptide
nanoparticle (SAPN).

[0046]In yet another preferred embodiment, one oligomerization domain D1
or D2 is the trimerization domain (foldon) of the bacteriophage T4
protein fibritin (Tao, Y. et al., Structure 1997, 5:789-798) or a
derivative thereof. This trimerization domain (m or n=3) has the sequence
GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:3). Small modifications of this
domain are also envisaged. Such modifications may be the substitution of
Asp 9 by Cys for the purpose of the formation of a disulfide bridge
between adjacent domains. Other modifications of surface amino acids of
this domain may include substitutions of residues for optimizing the
interactions at the interface between adjacent oligomerization domains
such as hydrophobic, hydrophilic or ionic interactions or covalent bonds
like disulfide bridges. Yet other modifications of surface amino acids of
this domain may include substitutions of amino acids (e.g. by cysteine or
lysine) for the generation of attachment sites for functional groups.

[0047]Most preferred are the coiled-coil sequences and monomeric building
blocks described in the examples.

Self-Assembling Peptide Nanoparticles: Even Units

[0048]Self-assembling peptide nanoparticles (SAPN) are formed from
monomeric building blocks of formula (I). If such building blocks
assemble, they will form so-called "even units". The number of monomeric
building blocks, which will assemble into such an even unit will be
defined by the least common multiple (LCM). Hence, if for example the
oligomerization domains of the monomeric building block form a trimer
(D1)3 (m=3) and a pentamer (D2)5 (n=5), 15 monomers will form
an even unit (FIG. 2A). If the linker segment L has the appropriate
length, this even unit may assemble in the form of a spherical peptidic
nanoparticle. Similarly, if the oligomerization domains D1 and D2 of the
monomeric building block form a trimer (D1)3 (m=3) and a tetramer
(D2)4 (n=4), the number of monomers needed to form an even unit will
be 12 (FIG. 2B).

[0049]Since m and n cannot be equal or a multiple of each other, the least
common multiple (LCM) is always larger than m and n.

[0050]Self-assembling peptide nanoparticles (SAPN) may be formed by the
assembly of only one or more than one even units (Table 2). Such SAPN
represent topologically closed structures.

[0051]There exist five regular polyhedra, the tetrahedron, the cube, the
octahedron, the dodecahedron and the icosahedron. They have different
internal rotational symmetry elements. The tetrahedron has a 2-fold and
two 3-fold axes, the cube and the octahedron have a 2-fold, a 3-fold and
a 4-fold rotational symmetry axis, and the dodecahedron and the
icosahedron have a 2-fold, a 3-fold and a 5-fold rotational symmetry
axis. In the cube the spatial orientation of these axes is exactly the
same as in the octahedron, and also in the dodecahedron and the
icosahedron the spatial orientation of these axes relative to each other
is exactly the same. Hence, for the purpose of SAPN of the invention the
cube and the octahedron, and similarly the dodecahedron and the
icosahedron can be considered to be identical. The cube/octahedron is
built up from 24 identical three-dimensional building blocks, while the
dodecahedron/icosahedron is built up from 60 identical three-dimensional
building blocks (Table 2). These building blocks are the asymmetric units
(AUs) of the polyhedron. They are tri-pyramids and each of the pyramid
edges corresponds to one of the rotational symmetry axes, hence these AUs
will carry at their edges 2-fold, 3-fold, and 4-fold or 5-fold symmetry
elements depending on the polyhedron type. If these symmetry elements are
generated from peptidic oligomerization domains such AUs are constructed
from monomeric building blocks as described above. It is sufficient to
align the two oligomerization domains D1 and D2 along two of the symmetry
axes of the AU (FIG. 3). If these two oligomerization domains form stable
oligomers, the symmetry interface along the third symmetry axis will be
generated automatically, and it may be stabilized by optimizing
interactions along this interface, e.g. hydrophobic, hydrophilic or ionic
interactions, or covalent bonds such as disulfide bridges.

[0052]To generate self-assembling peptide nanoparticles (SAPN) with a
regular geometry (dodecahedron, cube), more than one even unit is needed.
E.g. to form a dodecahedron from a monomer containing trimeric and
pentameric oligomerization domains, 4 even units, each composed of 15
monomeric building blocks are needed, i.e. the peptidic nanoparticle with
regular geometry will be composed of 60 monomeric building blocks. The
combinations of the oligomerization states of the two oligomerization
domains needed and the number of even units to form any of the regular
polyhedra are listed in Table 2.

[0053]Whether the even units will further assemble to form regular
polyhedra composed of more than one even unit depends on the geometrical
alignment of the two oligomerizations domains D1 and D2 with respect to
each other, especially on the angle between the rotational symmetry axes
of the two oligomerization domains. This is governed by i) the
interactions at the interface between neighboring domains in a
nanoparticle, ii) the length of the linker segment L, iii) the shape of
the individual oligomerization domains. This angle is larger in the even
units compared to the arrangement in a regular polyhedron. Also this
angle is not identical in monomeric building blocks as opposed to the
regular polyhedron. If this angle is restricted to the smaller values of
the regular polyhedron (by means of hydrophobic, hydrophilic or ionic
interactions, or a covalent disulfide bridge) and the linker segment L is
short enough, a given number of topologically closed even units each
containing a defined number of monomeric building blocks will then
further anneal to form a regular polyhedron (Table 2), or enclose more
monomeric building blocks to from nanoparticles lacking strict internal
symmetry of a polyhedron.

[0054]If the angle between the two oligomerization domains is sufficiently
small (even smaller than in a regular polyhedron with icosahedral
symmetry), then a large number (several hundred) peptide chains can
assemble into a peptidic nanoparticle. This can be achieved by replacing
the two cysteine residues that are located at the interface between the
two helices as in the original design of Raman S. et al., Nanomedicine:
Nanotechnology, Biology, and Medicine 2006, 2:95-102, and that are
forming a disulfide bridge between the two helices, by the small residue
alanine as in sequence SEQ ID NO:33. The angle between the two helices
can get smaller and consequently more than 60 peptide chains can assemble
into a SAPN. In such a design the SAPN have a molecular weight of about 4
MD, corresponding to about 330 peptide chains (Example 6).

T-Cell Epitopes and B-Cell Epitopes

[0055]Since the T-cell epitopes--as opposed to the B-cell epitopes--do not
need to be displayed on the surface of a carrier to cause immunization,
they can be incorporated into the core scaffold of the SAPN, i.e. the
coiled-coil sequence of an oligomerization domain. In the present
invention it is shown how the features of MHC binding of T-cell epitopes,
which requires an extended conformation for MHC binding (FIG. 1), can be
combined with the features of coiled-coil formation, which requires
α-helical conformation for coiled-coil formation, such that these
epitopes can be both, part of the coiled-coil scaffold of the SAPN as
well as being able to bind to the respective MHC molecules. It should be
noted that not all coiled-coil sequences will be able to bind to MHC
molecules and not all T-cell epitopes can be incorporated into a
coiled-coil structure. This invention provides the general rules, how to
select appropriate T-cell epitopes and describes the way how to
incorporate them into a particular coiled-coil oligomerization domain
such that these peptides will form SAPN. By using these rules a wide
variety of T-cell epitopes can be incorporated into the coiled-coil
scaffold of the SAPN.

[0056]In a further aspect of this invention B-cell epitopes that are not
coiled-coils are incorporated into the coiled-coil sequence of the SAPN
oligomerization domain by inserting them between two stretches of
coiled-coil segments, such that this whole sequence acts as a single
oligomerization domain. This is of particular interest as the coiled-coil
scaffold can provide means to restrict the conformation of the B-cell
epitope to a conformation that is nearly identical to its native
conformation.

Sources of T-Cell Epitopes

[0057]To incorporate T-cell epitopes into an oligomerization domain
leading finally to a self-assembling peptide nanoparticle (SAPN), the
T-cell epitopes can be chosen from different sources: For example, the
T-cell epitopes can be determined by experimental methods, they are known
from literature, they can be predicted by prediction algorithms based on
existing protein sequences of a particular pathogen, or they may be de
novo designed peptides or a combination of them.

[0058]There is a wealth of known T-cell epitopes available in the
scientific literature. These T-cell epitopes can be selected from a
particular pathogen (e.g. as in Examples 12, 13 and 14), from a cancer
specific peptide sequence (e.g. as in Example 4), or they may be de novo
designed peptides with a particular feature, e.g. the PADRE peptide (U.S.
Pat. No. 5,736,142) that binds to many different MHC II molecules, which
makes it a so-called promiscuous T-cell epitope (e.g. as in Example 1).
There exist commonly accessible databases that contain thousands of
different T-cell epitopes, for example the MHC-database "MHCBN VERSION
4.0" (http://www.imtech.res.in/raghava/mhcbn/index.html) or the
PDB-database "Protein Data Bank" (http://www.rcsb.org/pdb), or others.

[0059]It is well known and well documented that incorporation of HTL
epitopes into an otherwise not immunogenic peptide sequence or attaching
it to a non-peptidic antigen can make those much more immunogenic. The
PanDR binding peptide HTL epitope PADRE has widely been used in vaccine
design for a malaria, Alzheimer and many others vaccines.

[0060]According to the definition of the MHCBN database (supra) T-cell
epitopes are peptides that have binding affinities (IC50 values) of
less than 50,000 nM to the corresponding MHC molecule. Such peptides are
considered as MHC binders. According to this definition, as of August
2006, in the Version 4.0 of the MHCBN database the following data is
available: 20717 MHC binders and 4022 MHC non-binders.

[0061]Suitable T-cell epitopes can also be obtained by using prediction
algorithms. These prediction algorithms can either scan an existing
protein sequence from a pathogen for putative T-cell epitopes, or they
can predict, whether de novo designed peptides bind to a particular MHC
molecule. Many such prediction algorithms are commonly accessible on the
internet. Examples are SVRMHCdb (http://svrmhc.umn.edu/SVRMHCdb; J. Wan
et al., BMC Bioinformatics 2006, 7:463), SYFPEITHI
(http://www.syfpeithi.de), MHCPred (http://www.jenner.ac.uk/MHCPred),
motif scanner (http://hcv.lanl.gov/content/immuno/motif_scan/motif_scan)
or NetMHCIIpan (http://www.cbs.dtu.dk/services/NetMHCIIpan) for MHC II
binding molecules and NetMHCpan
(http://www.cbs.dtu.dk/services/NetMHCpan) for MHC I binding epitopes.

[0062]HTL epitopes as described herein and preferred for the design are
peptide sequences that are either measured by biophysical methods or
predicted by NetMHCIIpan to bind to any of the MHC II molecules with
binding affinities (IC50 values) better than 500 nM. These are
considered weak binders. Preferentially these epitopes are measured by
biophysical methods or predicted by NetMHCIIpan to bind to the MHC II
molecules with IC50 values better than 50 nM. These are considered
strong binders.

[0063]CTL epitopes as described herein and preferred for the design are
peptide sequences that are either measured by biophysical methods or
predicted by NetMHCpan to bind to any of the MHC I molecules with binding
affinities (IC50 values) better than 500 nM. These are considered
weak binders. Preferentially these epitopes are measured by biophysical
methods or predicted by NetMHCpan to bind to the MHC I molecules with
IC50 values better than 50 nM. These are considered strong binders.

Places for T-Cell Epitopes

[0064]The T-cell epitopes can be incorporated at several places within the
peptide sequence of the coiled-coil oligomerization domains D1 and or D2.
To achieve this, the particular sequence with the T-cell epitope has to
obey the rules for coiled-coil formation as well as the rules for MHC
binding. The rules for coiled-coil formation have been outlined in detail
above. The rules for binding to MHC molecules are incorporated into the
MHC binding prediction programs that use sophisticated algorithms to
predict MHC binding peptides.

[0065]There are many different HLA molecules, each of them having a
restriction of amino acids in their sequence that will best bind to it.
The binding motifs are summarized in Table 3. In this table the motif
shows x for positions that can have any amino acid, and in square
brackets the (list of) amino acids that can only be at a particular
position of the binding motif.

[0067]The frequency of occurrence of a particular amino acid at a certain
position of the T-cell epitope can also be summarized. For MHC binding
the positions 1, 4, 6 and 9 in a T-cell epitope are the most critical
ones. The most preferred residues at these positions are listed in Table
5, however, the preferences for particular amino acids at these position
vary largely between the different MHC molecules. Therefore, as mentioned
above, binding of a particular amino acid sequence to a MHC molecule can
be much more accurately predicted by the prediction programs listed
above.

[0068]From this Table 5 it is easily visible that, for example, the most
frequently encountered amino acids at position 1 and position 4 are the
ones that are found at core positions of the coiled-coil heptad repeat
(indicated by underlining). Position 1 and 4 can be superposed on the
heptad repeat positions aa(a) and aa(d). Therefore, a T-cell epitope with
the amino acid L in position 1 and amino acid V in position 4 is
perfectly in agreement with a coiled-coil peptide having the same amino
acids at the core positions aa(a) and aa(d) of the heptad repeat.
Therefore, if a peptide sequence obeys both, T-cell binding motif
restriction as well as coiled-coil heptad repeat motif restriction, it
can be incorporated into the coiled-coil oligomerization domain of the
SAPN. This can be achieved for a large number of T-cell epitopes by
adjusting the alignment of peptide sequence such that the T-cell binding
motif overlaps with the coiled-coil forming motif.

Engineering T-Cell Epitopes into Coiled-Coil

[0069]To engineer SAPN that incorporate T-cell epitopes in the coiled-coil
oligomerization domain of the SAPN, three steps have to be taken. In a
first step a candidate T-cell epitope has to be chosen by using known
T-cell epitopes from the literature or from databases or predicted T-cell
epitopes by using a suitable epitope prediction program. In a second step
a proteasomal cleavage site has to be inserted at the C-terminal end of
the CTL epitopes. This can be done by using the prediction program for
proteasomal cleavage sites PAProc
(http://www.paproc2.de/paproc1/paproc1.html; Hadeler K. P. et al., Math.
Biosci. 2004, 188:63-79) and modifying the residues immediately following
the desired cleavage site. This second step is not required for HTL
epitopes. In the third and most important step the sequence of the T-cell
epitope has to be aligned with the coiled-coil sequence such that it is
best compatible with the rules for coiled-coil formation as outlined
above. Whether the sequence with the incorporated T-cell epitope will
indeed form a coiled-coil can be predicted, and the best alignment
between the sequence of the T-cell epitope and the sequence of the
coiled-coil repeat can be optimized by using coiled-coil prediction
programs such as COILS (http://www.ch.embnetorg/software/COILS_form.html;
Gruber M. et al., J. Struct. Biol. 2006, 155(2):140-5) or MULTICOIL
(http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi), which
are available on the internet.

[0070]Even if it is not possible to find a suitable alignment--maybe
because the T-cell epitope contains a glycine or even a proline which is
not compatible with a coiled-coil structure--the T-cell epitope may be
incorporated into the oligomerization domain (see Example 3). In this
case the T-cell epitope has to be flanked by strong coiled-coil forming
sequences of the same oligomerization state. This will either stabilize
the coiled-coil structure to a sufficient extent or alternatively it can
generate a loop structure within this coiled-coil oligomerization domain.
This is essentially the same procedure as described in the next section
for the incorporation of B-cell epitopes into the coiled-coil core
sequence of the SAPN.

Engineering B-Cell Epitopes into the Coiled-Coil Core

[0071]In a particular aspect of this invention the incorporation into the
coiled-coil core of the SAPN of small B-cell epitopes that are not
α-helical is envisaged. This can be accomplished by the same
procedure as outlined above for the T-cell epitopes that are not
compatible with a coiled-coil structure. The structure of T4 fibritin
(pdb accession code 1aa0 at http://www.rcsb.org/pdb/) contains two loops
structures within its coiled-coil. The loops protrude from the
coiled-coil helix between two helical turns such that the helical
structure of the coiled-coil is not interrupted.

[0072]In fibritin, the loops leave the helix at position aa(b) of the
coiled-coil and reenter the helix at position aa(c) of the coiled-coil
sequence. One of the loops is a short beta-turn while the other is a more
irregular loop structure. The spacing between the residues aa(b) and
aa(c) in a coiled-coil is ideally suited to serve as anchor points for an
antiparallel beta-turn peptide. When residues aa(b) or aa(c) or both of
them are glycine residues this allows for the needed flexibility of the
protein secondary structure to exit and reenter the alpha-helix of the
coiled-coil.

TABLE-US-00006
(SEQ ID NO: 4)
..VQNLQVEIGNNSAGIKGQVVALNTLVNGTNPNGSTVEERGLTNSIKANETNIASVTQEV...
a d a d a d a d a d a d a

[0073]In the sequence of fibritin above the loops structures are displayed
in italic and the residues at the aa(b) and aa(c) positions (where the
two loops exit and reenter the helix) are indicated by underscores. Three
of these four residues are glycine residues. Taking this as a template, a
B-cell epitope that has an anti-parallel beta-turn conformation can now
be incorporated into the coiled-coil core of the SAPN. The coiled-coil
structure has to be sufficiently stable to allow incorporation of such a
loops structure, hence it must be able to form coiled-coils on both sides
of the loop. The smallest autonomously folding coiled-coil sequence
described so far is two heptad repeats long. In the sequence below the
tip of the V3 epitope from the protein gp120 of HIV, which is an
anti-parallel beta-turn peptide, is incorporated into the coiled-coil of
a designed stable coiled-coil with flanking helices of more than two
heptad repeats on both sides. These are very stable coiled-coil fragments
derived from Burkhard P. et al., J Mol Biol 2002, 318:901-910.

TABLE-US-00007
(SEQ ID NO: 5)
LEELERRLEELERRLEELERRLGSIRIGPGQTFYAGVDLELAALRRRLEELAR
a d a d a d a d a d a d core residues

[0074]This will restrict the conformation of the V3 loop within the
coiled-coil to an anti-parallel beta-turn conformation which corresponds
to the native conformation of this peptide on the protein.

Preferred Design

[0075]To engineer a SAPN with the best immunological profile for a given
particular application the following consideration have to be taken into
account:

[0076]CTL epitopes require a proteasomal cleavage site at their C-terminal
end. The epitopes should not be similar to human sequences to avoid
autoimmune responses--except when it is the goal to elicit an immune
response against a human peptide. Possible examples are the
cancer-specific CTL epitopes of Example 4.

[0077]Accordingly a SAPN is preferred wherein at least one of the T-cell
epitopes is a CTL epitope, and, in particular, wherein the sequence
further contains a proteasomal cleavage site after the CTL epitope.

[0078]Likewise preferred is a SAPN wherein at least one of the T-cell
epitopes is a HTL epitope, in particular, a pan-DR-binding HTL epitope.
Such pan-DR-binding HTL epitopes bind to many of the MHC class II
molecules as listed at the bottom of Table 3 and are therefore recognized
in a majority of healthy individuals, which is critical for a good
vaccine.

[0079]Also preferred is a SAPN wherein the sequence D1-L-D2 contains a
series of overlapping T-cell epitopes, either if D1 or D2 are a trimer
(Examples 7 and 8), a tetramer (Examples 9) or a pentamer (Examples 10).

[0080]B-cell epitopes need to be displayed at the surface of the SAPN.
They may or may not be part of the coiled-coil sequence, i.e. the
coiled-coil itself may partially be a B-cell epitope depending on whether
the portion of the coiled-coil is surface accessible. For example the
B-cell epitope composed of the trimeric coiled-coil of the surface
proteins of enveloped viruses can be displayed on the surface of the SAPN
and be part of coiled-coil sequence at the same time. An example of such
a design is presented in Raman S. et al., Nanomedicine: Nanotechnology,
Biology, and Medicine 2006; 2:95-102. Coiled-coils of any oligomerization
state in general are exceptionally well-suited to be presented in
conformation specific manner by the SAPN. Coiled-coils are abundant, not
only in enveloped virus surface proteins but also, for example, in the
genome of the malaria pathogen Plasmodium falciparum (Villard V. et al.,
PLoS ONE 2007; 2(7):e645).

[0081]In general, however, the B-cell epitopes will not be part of the
coiled-coil oligomerization domains, or they may be composed of a
coiled-coil and an additional portion that is not a coiled-coil, as for
example the trimeric autotransporter adhesions (TAA) of bacteria, which
have a coiled-coil stalk and a globular head domain, such as the TAA of
N. meningitidis. Of particular interest are proteins as B-cell epitopes
that are themselves oligomeric, such as trimeric hemagglutinin, and the
tetrameric sialidase or M2 surface proteins of influenza.

Considerations for the Design of a Vaccine Against a Pathogen

[0082]Such a vaccine preferably contains all three types of epitopes,
B-cell, HTL and CTL epitopes. (1) Preferably only one (or very few)
B-cell epitope should be placed at either end of the peptide chains. This
will place the B-cell epitope on the surface of the SAPN in a repetitive
antigen display. (2) The HTL epitopes should be as promiscuous as
possible. They do not necessarily need to be derived from the pathogen
but can be peptides that elicit a strong T-help immune response. An
example would be the PADRE peptide. Preferably these are the T-cell
epitopes that are incorporated into the D1-L-D2 core sequence of the
SAPN. (3) The CTL epitopes need to be pathogen specific, they need to
have C-terminal proteasomal cleavage sites. Since the T-cell epitopes do
not require repetitive antigen display several different T-cell epitopes
can be incorporated into one single SAPN by co-assembly of different
peptide chains that all have the same nanoparticle forming D1-L-D2 core
but carry different T-cell epitopes that are not part of the core forming
sequence and hence would not be incorporated into the coiled-coil
sequences.

[0083]In a similar manner peptide chains carrying an ER-targeting signal,
i.e. a signal peptide that induces the transport of a protein or peptide
to the endoplasmic reticulum (ER), can be co-assembled into the same SAPN
to bring the CTL epitopes into the ER for proper presentation by the MHC
I molecules since cross-presentation is not very efficient in humans. The
ER targeting signal, however, does not need to be on a separate peptide
chain, it can be in the same peptide as the CTL epitopes. A suitable ER
signal peptide would be for example the ER targeting signal (E3/19K)
MRYMILGLLALAAVCSA (SEQ ID NO:6).

Therapeutic Vaccine Aimed to Generate a Strong Antibody Response

[0084]A therapeutic vaccine aimed to generate a strong antibody response
is particularly useful for the treatment of Alzheimer, hypertension,
obesity, drug addictions, or inflammation. For such a vaccine preferably
only one B-cell epitope is used. The strong humoral immune response due
to repetitive antigen display can be further enhanced by including one or
more promiscuous HTL epitopes into the SAPN. Preferably these are the
T-cell epitopes that are incorporated into the D1-L-D2 core sequence of
the SAPN. Furthermore there should be as few and as weakly binding CTL
epitopes as possible--in particular not against a human peptide to avoid
autoimmune responses.

Therapeutic Vaccine to Induce a CTL Response, e.g. Against Cancer

[0085]In this case no B-cell epitope has to be used. The immune response
against the particular CTL epitopes (for example MAGE-1,2,3; MART-1,2,3;
or Her-2/neu, see also Example 4) is further enhanced by including one or
more promiscuous HTL epitopes into the SAPN.

Self-Assembling Peptide Nanoparticles (SAPN) as Adjuvants

[0086]A SAPN that is composed of many HTL epitopes will induce a strong
T-help immune response (see Example 2). If given in the same dose with
any other vaccine formulation this will result in a stimulation of the
immune response. Such a SAPN will be an adjuvant without the need of any
CTL or B-cell epitope. However, B-cell and CTL epitopes can be combined
with such an adjuvant SAPN. In addition particular adjuvant molecules can
be covalently coupled to the SAPN as a substituent to the oligomerization
domain D1 or D2 to further stimulate the adjuvant effect of the SAPN. Of
particular interest are immunostimulatory nucleic acids, preferably an
oligodeoxynucleotide containing deoxyinosine, an oligodeoxynucleotide
containing deoxyuridine, an oligodeoxynucleotide containing a CG motif,
an inosine and cytidine containing nucleic acid molecule. Other
immunostimulatory molecules are, for example, antimicrobial peptides such
as cationic peptides which are a class of immunostimulatory, positively
charged molecules that are able to facilitate and/or improve adaptive
immune responses. An example of such a peptide with immunopotentiating
properties is the positively charged artificial antimicrobial peptide
KLKLLLLLKLK (SEQ ID NO: 63), which induces potent protein-specific type-2
driven adaptive immunity after prime-boost immunizations.

[0087]Preferably, antigens of the invention are selected from the group
consisting (a) proteins suited to induce an immune response against
cancer cells; (b) proteins or carbohydrates suited to induce an immune
response against infectious diseases; (c) proteins suited to induce an
immune response against allergens; (d) peptide hormones suited to induce
an immune response for the treatment of a human disease; and (e) hapten
molecules suited to induce an immune response to treat addictions or
other disorders. Peptidic nanoparticles comprising such proteins,
peptidic fragments thereof, peptides, carbohydrates, or haptens may be
suited to induce an immune response in humans, or also in farm animals
and pets.

[0088]In one preferred embodiment of the invention, the antigens or
antigenic determinants are the ones that are useful for the prevention of
infectious disease. Such treatment will be useful to prevent a wide
variety of infectious diseases affecting a wide range of hosts, e.g.
humans or non-human animals, such as cow, sheep, pig, dog, cat, other
mammalian species and non-mammalian species as well.

[0089]In particular the invention relates to SAPN comprising one of the
following antigens:

(a) an antigen suited to induce an immune response against bacteria;(b) an
antigen suited to induce an immune response against viruses;(c) an
antigen suited to induce an immune response against parasites;(d) an
antigen suited to induce an immune response against cancer cells;(e) an
antigen suited to induce an immune response against allergens;(f) an
antigen suited to induce an immune response against addictions;(g) an
antigen suited to induce an immune response against diseases and
metabolic disorders;(h) an antigen suited to induce an immune response in
a farm animals; and(i) an antigen suited to induce an immune response in
a pet.

[0090]Treatable infectious diseases are well known to those skilled in the
art. Examples include infections of viral, bacterial or parasitic
etiology such as the following diseases:

[0092]In a preferred aspect of the invention, a composition for the
prevention and treatment of malaria is envisaged (Example 11). The life
cycle of the malaria parasite provides several stages at which
interference could lead to cessation of the infective process. In the
life cycle of the malaria parasite, a human becomes infected with malaria
from the bite of a female Anopheles mosquito. The mosquito inserts its
probe into a host and in so doing, injects a sporozoite form of
Plasmodium falciparum (or vivax), present in the saliva of the mosquito.
Possible protein and peptide sequences suitable for the design of a
peptide vaccine may contain sequences from the following Plasmodium
proteins: MSP-1 (a large polymorphic protein expressed on the parasite
cell surface), MSA1 (major merozoite surface antigen 1), CS protein
(native circumsporozoite), 35 KD protein or 55 KD protein or 195 KD
protein according to U.S. Pat. No. 4,735,799, AMA-1 (apical membrane
antigen 1), or LSA (liver stage antigen).

[0093]In a preferred design one of the B-cell epitopes is a sequence of 8
to about 48 residues that constitute a B cell epitope of the
circumsporozoite (CS) protein. This B-cell epitope is a redundant repeat
region of the amino acid sequence NANP for Plamodium falciparum. In a
preferred SAPN design this B cell epitope comprises two to about five
repeats of the amino acid residue sequence NANP or the permutations
thereof ANPN, NPNA, and PNAN. The corresponding repeat region in
Plasmodium vivax is composed of any of the following highly similar
sequences

[0094]In a preferred design one of the B-cell epitopes is a sequence of 8
to about 48 residues composed of any of these sequences.

[0095]Specific peptide sequences for the design of SAPN for the treatment
of malaria are listed in the three tables below for B-cell epitopes,
HTL-epitopes and CTL-epitopes.

[0096]The following Table 7 lists preferred P. falciparum coiled-coil
B-cell epitopes (Villard V. et al., PLoS ONE 2007, 2(7):e645 and Agak G.
W., Vaccine (2008) 26, 1963-1971. Since for B-cell epitopes only the
surface accessible residues are of critical importance for their
interactions with the B-cell receptor and the production of antibodies,
the coiled-coil core residues at aa(a) and aa(d) positions, which are not
surface exposed can be modified to some extent without changing the
ability of the immunogen to elicit neutralizing antibodies. For example,
exchanging a valine at an aa(a) position with an isoleucine will not
affect the general immunological properties of the coiled-coil B-cell
epitope. Therefore these coiled-coil sequences can be artificially
stabilized by optimizing the core residues for best coiled-coil formation
and stability (Example 13) without abolishing their immunological
potential. Accordingly, modifications of these peptide B-cell epitopes at
one or more of their core residues at aa(a) and/or aa(d) in line with the
coiled-coil forming propensities as outlined in detail above are also
envisioned for these B-cell epitopes.

[0097]Thus, in a preferred design, the coiled-coil B-cell epitope with
modifications of one or more of their core positions is a peptide that
contains at least a sequence which is two heptad-repeats long that is
predicted by the coiled-coil prediction program COILS to form a
coiled-coil with higher probability than 0.9 for all its amino acids with
at least one of the window sizes of 14, 21, or 28.

[0098]In a more preferred design, the coiled-coil B-cell epitope with
modifications of one or more of their core positions is a peptide that
contains at least one sequence three heptad-repeats long that is
predicted by the coiled-coil prediction program COILS to form a
coiled-coil with higher probability than 0.9 for all its amino acids with
at least one of the window sizes of 14, 21, or 28.

[0099]In another more preferred design, the coiled-coil B-cell epitope
with modifications of one or more of their core positions is a peptide
that contains at least two separate sequences two heptad-repeats long
that are predicted by the coiled-coil prediction program COILS to form a
coiled-coil with higher probability than 0.9 for all its amino acids with
at least one of the window sizes of 14, 21, or 28.

[0102]In another preferred aspect of the invention, a composition for the
prevention and treatment of HIV is envisaged (Examples 5 and 12). For the
preparation of an anti-HIV vaccine a synthetic peptide capable of
eliciting HIV-specific antibodies may be used, said synthetic peptide
having the amino acid sequence of a functional T-cell epitope or B-cell
epitope of an envelope or gag protein or gp120 or gp41 of HIV-1 to
provide an immune response. Of special interest are sequences within gp41
or gp120, which can induce conformation specific neutralizing antibodies
able to interfere with the fusion process like the known antibodies 2F5
and 4E10 or from the V3-loop of gp41 or gp120. Such sequences are mainly
localized in and around the HR1 and HR2 and the cluster I and cluster II
regions. Antibodies binding to e.g. the coiled-coil trimer of gp41 and
elicited by peptidic nanoparticles of the invention incorporating this
coiled-coil trimer will inhibit hairpin formation and hence viral fusion.
Similarly, antibodies raised against the trimeric coiled-coil of Ebola or
of another virus with a similar fusion process will inhibit viral entry
of these viruses.

[0103]Using the highly conserved HIV protein sequences as described in
Letourneau S. et al., PLoS ONE 2007, 10:e984, CTL epitopes were predicted
using SVRMHCdb (http://svrmhc.umn.edu/SVRMHCdb; Wan J. et al., BMC
Bioinformatics 2006, 7:463). These conserved protein sequences contain
CTL epitopes predicted to bind to the HLA molecules as listed in Table 10
and are preferred CTL epitopes for the design of an SAPN-HIV vaccine.
These peptide epitopes contain largely overlapping sequences that can be
combined to longer peptide sequences that harbor multiple CTL epitopes in
one single continuous peptide string (Table 11).

[0104]In another preferred aspect of the invention, a composition for the
prevention and treatment of influenza is envisaged. Influenza A encodes
an integral membrane protein, M2, a homotetramer, the subunit of which
has a small external domain (M2e) of 23 amino acid residues. The natural
M2 protein is present in a few copies in the virus particle and hidden to
the immune system by the bulky other two surface proteins hemagglutinin
and sialidase. On the other hand it exists in abundance on the membrane
surface of virus-infected cells. The sequence of M2e is highly conserved.
It has been shown that M2e presented to the immune system as a tetramer
in a chimeric GNC4-M2e protein, generates a highly specific and
protective humoral immune response (DeFilette M. et al., J Biol Chem
2008; 283(17):11382-11387).

[0105]The M2e tetramer is a highly conserved B-cell epitope for both,
human and avian specific influenza strains (Table 12 and 13). In a
preferred embodiment of this invention it can be displayed in its native
tetrameric conformation when attached to the N-terminus of the tetrameric
coiled-coil from tetrabrachion--or any other tetrameric coiled-coil--of
the SAPN (Example 9).

[0106]Influenza hemagglutinin (HA) is activated by cleavage of the
precursor protein into two separate peptide chains (Steinauer D. S. et
al., Virology 1999; 258:1-20). Cleavage of the HA precursor molecule HA0
is required to activate virus infectivity, and the distribution of
activating proteases in the host is one of the determinants of tropism
and, as such, pathogenicity. The HAs of mammalian and nonpathogenic avian
viruses are cleaved extracellularly, which limits their spread in hosts
to tissues where the appropriate proteases are encountered. On the other
hand, the HAs of pathogenic viruses are cleaved intracellularly by
ubiquitously occurring proteases and therefore have the capacity to
infect various cell types and cause systemic infections.

[0107]In contrast to the M2e sequence, the N-terminal part of the cleavage
peptide is not highly conserved (the C-terminal portion of the cleavage
peptide is in fact highly conserved). In the HA precursor protein the
cleavage peptide is surface exposed and the six residues (three residues
on each side of the cleavage site) around the cleavage site are the most
characteristic of this peptide sequence (highlighted in bold in Table
14). In a preferred SAPN design, these six residues represent the B-cell
epitope, which can induce antibodies that, upon binding to the peptide,
can protect the HA precursor protein from getting cleaved.

[0108]The SAPN are ideally suited to display a multitude of different
cleavage sequences specific for the different HA types (Table 14) by
co-assembling peptides that have the same SAPN-forming core D1-L-D2 but
different B-cell epitopes attached to it (Example 15).

[0109]For example a hemagglutinin B-cell epitope string comprising the
sequences of H1, H2, and H3 cleavage sites with an inserted aspartate
amino acid to make the sequence more soluble and less basic would look
like this: SIQSRGLFGDIESRGLFGERQTRGIFG (SEQ ID NO:227).

[0110]Peptides with the same core sequence but different B-cell epitopes
or epitope strings can be co-assembled into a single SAPN to generate a
multivalent SAPN immunogen that possibly includes all or the most
important (H1, H2, H3, H5, H7 and H9 for a human vaccine) sequences of
Table 14 (Example 15).

[0111]In a similar approach an Influenza vaccine SAPN composed of six
peptide chains with an identical core and identical N-terminal B-cell
epitope M2e and about 20 CTL epitopes at the C-terminus (three or four
each per peptide chain) can be co-assembled into a single SAPN (Example
14). In Table 15 preferred conserved CTL epitopes from Panda R. et al.,
Vaccine 2007, 25:7530-7539 are listed. Since the core of these six
peptide chains is identical, co-assembly of these six peptide chains into
one single SAPN allows the incorporation of about 20 different CTL
epitopes into one single SAPN.

[0112]In another preferred embodiment, the compositions of the invention
are immunotherapeutics that may be used for the treatment of metabolic
disorders and diseases, or addictions. Most preferred are
immunotherapeutics for the treatment of Alzheimer disease, hypertension,
obesity, nicotine- and cocaine addictions.

[0113]The Aβ-fragment (Aβ) is a 42 amino acid long peptide
(Aβ1-42). Since the whole 42 residue long peptide sequence also
contains CTL epitopes that can cause autoimmune reactions, it is
desirable to use only shorter fragments of this peptide for vaccine
design, such as Aβ1-12 or even such short peptides as Aβ1-6
(U.S. Pat. No. 7,279,165).

[0114]Likewise, the full-length TNFα protein as an immunogen has
some limitations. Local overproduction of the proinflammatory cytokine
TNFα is critically involved in the pathogenesis of several chronic
inflammatory disorders, including rheumatoid arthritis, psoriasis, and
Crohn's disease. Neutralization of TNFα by monoclonal antibodies
(mAbs, infliximab, adalimumab) or chimeric soluble receptors (etanercept)
is efficacious in the treatment of these conditions but has several
potential drawbacks. It may induce allotype- or Id-specific Abs, which
might limit long-term efficacy in many patients. Moreover, as the number
of treated patients increases, it is becoming evident, that treatment
with TNFα-antagonists, in particular mAbs, increases the risk of
opportunistic infections, especially those caused by intracellular
pathogens like Mycobacterium tuberculosis, Listeria monocytogenes, or
Histoplasma capsulatum. Immunization with shorter fragments of TNFα
comprising only residues 4-23 has been shown to avoid some of these
problems (G. Spohn et al., The Journal of Immunology, 2007, 178:
7450-7457). Therefore, immunization with TNFα4-23 is a novel
efficient therapy for rheumatoid arthritis and other autoimmune
disorders, which adds a new level of safety to the existing
anti-TNFα therapies. By selectively targeting only the soluble form
of TNFα and sparing the transmembrane form, pathogenic effects of
TNFα are neutralized by the vaccine, while important functions in
the host response to intracellular pathogens remain intact.

[0116]In another preferred embodiment, the compositions of the invention
are immuno-therapeutics that may be used for the treatment of allergies.
The selection of antigens or antigenic determinants for the composition
and the method of treatment for allergies would be known to those skilled
in the medical art treating such disorders. Representative examples of
this type of antigen or antigenic determinant include bee venom
phospholipase A2, Bet v I (birch pollen allergen), 5 Dol m V (white-faced
hornet venom allergen), and Der p I (House dust mite allergen).

[0118]The selection of antigens or antigenic determinants for the
composition and method of treatment for cancer would be known to those
skilled in the medical art treating such disorders. Representative
examples of this type of antigen or antigenic determinant include the
following: HER2/neu (breast cancer), GD2 (neuroblastoma), EGF-R
(malignant glioblastoma), CEA (medullary thyroid cancer), CD52
(leukemia), MUC1 (expressed in hematological malignancies), gp100
protein, or the product of the tumor suppressor gene WT1. In Table 17
cancer specific T-cell epitopes of interest are shown with the relevant
protein of origin and the MHC restriction.

[0119]Most preferred are the coiled-coil sequences, B-cell-, HTL- and
CTL-epitopes, monomeric building blocks, SAPN, and vaccine designs
described in the examples.

EXAMPLES

[0120]The following examples are useful to further explain the invention
but in no way limit the scope of the invention.

Example 1

PADRE (P5c-8-Mal)

[0121]The pan-DR epitope PADRE with the sequence AKFVAAWTLKAAA (SEQ ID
NO:296) is incorporated into the trimeric coiled-coil of the SAPN by
using the following design criteria: The residue alanine (A) has a strong
tendency to form alpha-helices. The alignment with coiled-coil core
positions is such that the residues valine, tryptophane and alanine are
at an aa(a), aa(d) and again an aa(a) position of the heptad repeat
pattern. The remaining part of the trimeric coiled-coil N-terminal and
C-terminal to the HTL epitope is designed such that the sequence is
predicted to from a very strong coiled-coil.

[0122]The sequence of this peptide (SEQ ID NO:7) is predicted to form a
coiled-coil by the prediction program COILS. The coiled-coil forming
probability is more than 95% for all the residues in the sequence using a
window of 21 amino acids for the coiled-coil prediction (see Table 18
below). Hence this is a predicted coiled-coil that also contains a T-cell
epitope.

[0123]It is very important to realize that if the window comprises only 14
amino acids, the coiled-coil prediction drops to very low values of less
than 2% probability within the sequence of the T-cell epitope. The larger
window size of 21 amino acids with its high predicted coiled-coil
propensities throughout the sequence shows the effect of the flanking
regions at the N- and C-terminal ends of the T-cell epitope. If these are
sequences that strongly favor coiled-coil formation then the less
favorable coiled-coil propensity of the T-cell epitope may be compensated
and the whole sequence will be induced to form a coiled-coil even though
the T-cell epitope does not contain a very favorable coiled-coil
sequence.

[0124]This trimeric coiled-coil of SEQ ID NO:7 was then included in the
SAPN sequence of SEQ ID NO:8 below, which is composed of a His-tag, the
pentameric coiled-coil of COMP, the trimeric coiled-coil composed of the
sequence of SEQ ID NO:7 comprising the PADRE T-cell epitope, and the
B-cell epitope from the CS-protein of Plasmodium berghei.

[0125]The peptide with this sequence was expressed in E. coli and purified
on a nickel affinity column by standard biotechnology procedures. The
refolding was performed according to Raman S. et al., Nanomedicine:
Nanotechnology, Biology, and Medicine 2 (2006) 95-102. The refolded SAPN
were analyzed for nanoparticle formation by dynamic light scattering
(DLS) techniques and transmission electron microscopy (TEM). The DLS
analysis showed a nice size distribution with an average particle
diameter of 32.01 nm and polydispersity index of 12.9% (FIG. 4A). The TEM
pictures (FIG. 4B) show nanoparticles of the same size as determined by
DLS.

Example 2

P5c-6-General

[0126]This coiled-coil sequence contains four overlapping HTL epitopes
with the sequences LEELERSIW, IWMLQQAAA, WMLQQAAAR, and MLQQAAARL that
are predicted by the algorithm SVRMHC to bind to the different MHC II
molecules DQA1*0501, DRB1*0501, DRB5*0101, and DRB1*0401 respectively,
with predicted binding affinities (pIC50 values) of 6.122, 8.067, 6.682,
and 6.950 respectively. These HTL epitopes are aligned with the
coiled-coil heptad repeat such that they are predicted to form a very
strong coiled-coil. The aa(a) and aa(d) core positions of the coiled-coil
are occupied by Leu, Leu, Ile, Leu, Ala and Leu, most of them very good
residues for high coiled-coil forming propensity with only Ala being
somewhat less favorable.

[0127]Consequently the sequence of the peptide is predicted to form a
coiled-coil by the prediction program COILS. The coiled-coil forming
probability is more than 99% for all the residues of the HTL epitopes in
the sequence (Table 19). Comparing the small and large window sizes for
coiled-coil prediction shows again the influence of the flanking
sequences for the coiled-coil stability. With a window size of 28 amino
acids the whole sequence is predicted to form a coiled-coil with a
probability of 100% while the smaller window size of 14 amino acids shows
the effect of the lower coiled-coil propensity of the T-cell epitopes at
the N-terminal end with lower prediction values for coiled-coil
formation. Hence for the whole sequence this peptide is predicted to from
a stable coiled-coil including the four different HTL epitopes.

[0128]This trimeric coiled-coil was then included into the SAPN sequence
SEQ ID NO:10 below, composed of a His-tag, the pentameric coiled-coil of
COMP, the trimeric coiled-coil of SEQ ID NO:9 and the B-cell epitope from
the CS-protein of Plasmodium berghei.

[0129]The peptide with this sequence was expressed in E. coli and purified
on a nickel affinity column by standard biotechnology procedures. The
refolding was performed according to Raman S. et al., Nanomedicine
(supra). The refolded SAPN were analyzed for nanoparticle formation by
dynamic light scattering (DLS) techniques and transmission electron
microscopy (TEM). The DLS analysis showed a nice size distribution with
an average particle diameter of 46.96 nm and very low polydispersity
index of 8.7%. The TEM pictures (FIG. 5) show nanoparticles of the size
of about 30 nm.

Example 3

T1c-7-Influenza

[0130]This coiled-coil sequence contains two consecutive HTL epitopes with
the sequences IRHENRMVL and YKIFKIEKG from the proteins M1 and
neuraminidase of the influenza A virus, respectively. By the computer
algorithm SVRMHC they are predicted to strongly bind to the MHC II
molecules DRB1*0405 and DRB1*0401 with predicted binding affinities
(pIC50 values) of 8.250 and 6.985, respectively. Furthermore, according
to Panda R. et al., Vaccine 2007, 25:7530-7539, the first T-cell epitope
IRHENRMVL is predicted to bind to many other HLA molecules as well such
as B14, B1510, B2705, B2706, B3909, DP9, DR11, DR12, DR17, DR53, and
DRB1, i.e. it is a predicted promiscuous epitope.

[0131]The best alignment of these HTL epitopes with the coiled-coil heptad
repeat is shown below (SEQ ID NO:11). However, the sequence of the second
of these epitopes contains an unfavorable glycine residue that in general
acts as a helix breaking residue. The flanking parts of the coiled-coil
trimer are relatively short sequences but have strong coiled-coil forming
propensity. When using a small window of 14 amino acids in the
coiled-coil prediction program COILS it is visible that coiled-coil
structures are predicted for both sides of the T-cell epitopes (see Table
20 below). Hence the whole sequence will again act as a single folding
unit and form a stable coiled-coil with a trimeric oligomerization state.

[0132]This trimeric coiled-coil was then included into the SAPN sequence
SEQ ID NO:12 below, composed of a His-tag, the pentameric Trp-zipper
coiled-coil, the trimeric coiled-coil as described above and the B-cell
epitope from the CS-protein of Plasmodium berghei.

[0133]The peptide with this sequence was expressed in E. coli and purified
on a nickel affinity column by standard biotechnology procedures. The
refolding was performed according to Raman S. et al., Nanomedicine
(supra). The refolded SAPN were analyzed for nanoparticle formation by
dynamic light scattering (DLS) techniques and transmission electron
microscopy (TEM). The DLS analysis showed a somewhat broader size
distribution with an average particle diameter of 57.70 nm and
polydispersity index of 21.6%. The TEM pictures (FIG. 6) show
nanoparticles of the size of about 30-50 nm. Some of them have the
tendency to stick together and the refolding conditions would need some
more improvement.

Example 4

Prediction of Coiled-Coils for Cancer CTL Epitopes

[0134]In the following examples it is shown how cancer specific T-cell
epitopes can be included into the SAPN forming peptide. 19 different
T-cell epitopes are engineered into the trimeric coiled-coil of the SAPN
and the corresponding coiled-coil propensities are calculated for the
first 10 of these sequences. It is nicely visible that the coiled-coil
propensities for the T-cell epitopes are rather low, but the flanking
sequences with very high coiled-coil propensities will compensate and
induce coiled-coil formation throughout the whole sequence similar as
shown for the examples 1 to 3 above.

[0135]The following sequences show likewise a high coiled coil propensity,
although the coiled-coil propensities for the T-cell epitopes are rather
low, but the flanking sequences with very high coiled-coil propensities
will compensate and induce coiled-coil formation throughout the whole
sequence.

[0136]This coiled-coil sequence contains the anti-parallel beta-turn
peptide, which is the tip of the V3 loop of gp120 from HIV. This peptide
is a well-known B-cell epitope of HIV. It is inserted into the
coiled-coil heptad repeat by two glycine residues at positions aa(b) and
aa(c) of the coiled-coil.

[0137]The flanking parts of the coiled-coil trimer are relatively short
sequences but have strong coiled-coil forming propensity. When using a
small window of 14 amino acids in the coiled-coil prediction program
COILS it is visible that coiled-coil structures are predicted for both
sides of the B-cell epitopes (see Table 22 below). Hence the whole
sequence will act as a single folding unit and form a stable coiled-coil
with a trimeric oligomerization state with a protruding beta-turn
peptide.

[0139]This sequence is related to the sequences from Examples 1 and 2 (SEQ
ID NO:8 and SEQ ID NO:10), but without the C-terminal B-cell epitope. The
SAPN formed do not have a disulfide bridge between the two helices
(underlined residues 55 and 64, replacement of the two cysteines by
alanine as compared to the original design of Raman S. et al.,
Nanomedicine: Nanotechnology, Biology, and Medicine 2006; 2:95-102) but
rather have the smaller amino acid alanine instead, allowing for smaller
angles between the two helices, and hence more than 60 peptide chains are
incorporated into the SAPN.

[0140]Three conditions were tested for assembling nanoparticles from the
monomeric building block SEQ ID NO:33. The molecular weight (MW) of the
SAPN was assessed by analytical ultracentrifugation: The peptide was
dissolved at 0.42 mg/ml, 0.34 mg/ml, and 0.21 mg/ml, in 150 mM NaCl, 20
mM Tris, pH 7.5. The measured MW corresponds to SAPN composed of about
330 monomers, i.e. a nanoparticle with more monomers than needed for a
regular polyhedron with 60 asymmetric units (Table 23). The two helices
of the two oligomerization domains are not fixed by a disulfide bridge in
their relative orientation to each other and the smaller amino acid
alanine allows the two helices to get closer and hence the angle between
them to be smaller.

Trimeric Coiled-Coil with a Series of Overlapping Measured HTL Epitopes
(pan3m)

[0141]The following is an example of a trimeric coiled-coil design that
includes peptide epitopes from Hepatitis B Virus polymerase, which have
been measured for their binding affinities to different MHCII molecules
(Mizukoshi E. et al., J Immunol 2004, 173:5863-5871). Two of these
peptide have sequentially been designed into the trimeric coiled coil
according to the principles outlined in this document.

TABLE-US-00036
RLLARLEELERRLEELQSLTNLLSSNLSWLSLDVSAAFRRLEELEARVM (SEQ ID NO: 34)
d a d a d a d a d a d a d a core residues

[0143]The sequence of the peptide is predicted to form a coiled-coil by
the prediction program COILS. The coiled-coil forming probability is more
than 98% for all the residues in the sequence (Table 24), therefore the
whole sequence is predicted to form a fully folded coiled-coil.

Trimeric Coiled-Coil with a Series of Overlapping Predicted HTL Epitopes
(pan3p)

[0144]The following is an example of a trimeric coiled-coil design that
includes a subsection of the HTL epitopes from SEQ ID NO:34 from
(Mizukoshi E. et al., J Immunol 2004, 173:5863-5871) in combination with
a sequence that is a subsection of the PADRE HTL epitope sequence.

[0145]The following are the binding affinities as predicted by the
algorithm NetMHCII of the epitopes contained in the sequence
ARFVAAWTLKVREVERELSWLSLDVSAAF for the different MHCII molecules as
follows (binding affinities in nM in brackets): DRB1*0101 (23), DRB1*0401
(72), DRB1*0405 (37), DRB1*0701 (164), DRB1*0802 (462), DRB1*0901 (440),
DRB1*1101 (271), DRB1*1302 (303), DRB1*1501 (16), DRB3*0101 (60),
DRB4*0101 (51). This sequence contains in some part the same epitopes as
the sequence pan3m (SEQ ID NO:34), which have in fact shown even stronger
binding to the MHCII molecules than predicted by the NetMHCII program
(Mizukoshi E. et al., J Immunol 2004, 173:5863-5871).

[0146]The sequence of the peptide is predicted to form a coiled-coil by
the prediction program COILS. The coiled-coil forming probability is more
than 80% for all the residues in the sequence (Table 25) except for the
last 8 residues, therefore the sequence is predicted to form a fully
folded coiled-coil with the C-terminal end fraying a little bit apart,
which will not interfere with SAPN formation as the majority of the
sequence at N-terminal end form a coiled-coil.

Tetrameric Coiled-Coil with a Series of Partly Overlapping Predicted HTL
Epitopes (BN5c-M2eN)

[0147]The tetrameric coiled-coil sequence from tetrabrachion (Stetefeld J.
et al., Nature Structural Biology 2000, 7(9):772-776) is characterized by
an undecad coiled-coil repeat rather than a heptad repeat. The following
is a slightly modified sequence derived from this tetrameric coiled-coil.

[0148]This tetrameric coiled-coil is particularly well-suited as core
coiled-coil of the SAPN as it contains a series of overlapping predicted
HTL epitopes. The sequences of the epitopes YRLTVIIDD, LKNLITLRA,
LITLRADRL, IINDNVSTLR, INDNVSTLRA, and VSTLRALLM are predicted by the
algorithm NetMHCII to bind to the different MHC II molecules DRB1*0101,
DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0701, DRB1*1101, DRB1*1302, and
DRB1*1501, respectively, with predicted binding affinities (nM) of 3, 48,
78, 162, 243, 478, 12, and 420 respectively.

[0149]The tetrameric coiled-coil is also well-suited to present tetrameric
B-cell epitopes such as the M2e peptide from influenza, which has been
done in the following SAPN design for a human vaccine with the sequence
(SEQ ID NO:37):

[0150]The peptide (SEQ ID NO:37) contains starting from the N-terminus:
the His-tag, the M2e B-cell epitope from a human-specific influenza
strain, the tetrameric coiled-coil with the HTL epitopes, the linker, and
the trimeric coiled-coil. With this sequence it was expressed in E. coli
and purified on a nickel affinity column by standard biotechnology
procedures. The refolding was performed according to Raman S. et al.,
Nanomedicine: Nanotechnology, Biology, and Medicine 2006; 2:95-102. The
refolded SAPN were analyzed for nanoparticle formation by transmission
electron microscopy (TEM). The TEM pictures (FIG. 7A) show nanoparticles
of the same of about 30 nm.

[0151]Also the chicken-specific M2e peptide from influenza can be
displayed in its native oligomerization state and conformation as
tetramer on the tetrameric coiled-coil of tetrabrachion, which has been
done in the following SAPN design for an animal vaccine with the sequence
(SEQ ID NO:38):

[0152]The peptide with this sequence (SEQ ID NO:38) was expressed in E.
coli and purified on a nickel affinity column by standard biotechnology
procedures. It contains starting from the N-terminus: the His-tag, the
M2e B-cell epitope from a chicken-specific influenza strain, the
tetrameric coiled-coil with the HTL epitopes, the linker, and the
trimeric coiled-coil. The refolding was performed according to Raman S.
et al., Nanomedicine: Nanotechnology, Biology, and Medicine 2006;
2:95-102 with the buffer of 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5%
glycerol. The refolded SAPN were analyzed for nanoparticle formation by
dynamic light scattering (DLS) techniques and transmission electron
microscopy (TEM). The DLS analysis showed a size distribution with an
average particle diameter of 45 nm and polydispersity index of 8.9%. The
TEM pictures (FIG. 7B) show nanoparticles of the same size of about 30
nm.

Example 10

Pentameric Coiled-Coil with a Series of Overlapping Predicted HTL Epitopes

[0153]This sequence is predicted to form an α-helix (H) with high
probability (mostly the highest score 9) according to the secondary
structure prediction program PSIPRED
(http://bioinf.cs.ucl.ac.uk/psipred/--The PSIPRED Protein Structure
Prediction Server). Since the core positions aa(a) and aa(d) of the
heptad repeat of the coiled-coil are mostly tryptophane residues, this
sequence is predicted to form a pentameric coiled coil (Liu J et al.,
Proc Natl Acad Sci USA 2004; 101(46):16156-61).

[0155]The following is an example of a trimeric coiled-coil design that
includes a pan DR binding epitope from Plasmodium falciparum. The
sequence corresponds to the 17 C-terminal amino acids with two cysteines
replaced by alanines, also know as CS.T3 peptide (SEQ ID NO:40) from the
circum sporozoite protein CS.

[0156]In a cell proliferation assay it has been shown that this CS.T3
peptide had pan DR activity and was stimulatory for DR1, DR2, DR4, DR5,
DRw6, DR7 and DR9 molecules (U.S. Pat. No. 5,114,713).

[0157]The sequence of the peptide is predicted to form a coiled-coil by
the prediction program COILS. The coiled-coil forming probability is more
than 99% for all the residues in the sequence (Table 26), except for the
last two amino acids. Therefore the whole sequence is predicted to form a
fully folded coiled-coil.

[0159]This sequence (SEQ ID NO 41) contains a his-tag, a pentameric
coiled-coil tryptophane zipper, a linker, the trimeric coiled-coil SEQ ID
NO:40, and a Plasmodium falciparum B-cell epitope, which is a
tetra-repeat (NANP) of the repetitive sequence of the same circum
sporozoite protein CS.

[0160]The peptide with this sequence was expressed in E. coli and purified
on a nickel affinity column by standard biotechnology procedures. The
refolding was performed according to Raman S. et al., Nanomedicine
(supra). The refolded SAPN were analyzed for nanoparticle formation by
dynamic light scattering (DLS) techniques and transmission electron
microscopy (TEM). The DLS analysis at pH 6.5 showed a size distribution
with an particle diameter of 44.6 nm and a polydispersity index of 19.6%.
The TEM pictures (FIG. 8) show nanoparticles of the size of about 30 nm.

Example 12

HIV Vaccine: HTL, CTL, B-Cell Co-Assembly

[0161]The following is an example of an HIV vaccine design. These
conserved protein sequences contain CTL epitopes predicted to bind to the
HLA molecules as listed in Table 10.

[0162]The first seven CTL peptide-strings of Table 10 are engineered at
the C-terminal end of the six peptide chains with identical core and
identical N-terminal B-cell epitope and hence are co-assembled into a
single SAPN.

[0163]The core contains the same trimer as in Example 11 with a
promiscuous P. falciparum HTL epitope, linked to a Trp-zipper pentameric
coiled-coil which contains a PADRE HTL epitope.

[0164]The B-cell epitope is the membrane proximal region of GP41, which
has neutralizing potential as evidenced by binding of the monoclonal
neutralizing antibodies 2F5 and 4E10. This epitope is α-helical in
solution and is held in α-helical conformation by being partly
engineered into the pentameric coiled-coil. In this design the surface
accessible residues (i.e. the residues that are not the coiled-coil core
residues) are the ones that are bound by the antibody 4E10.

Example 13

Malaria Vaccine: HTL-CTL-B-Cell-Core, B-Cell, CTL Co-Assembly

[0165]The following is an example of an malaria vaccine SAPN composed of
six peptide chains with an identical core and an identical C-terminal
B-cell epitope and about 18 CTL epitopes at the N-terminus (three each
per peptide chain) co-assembled into a single SAPN.

[0166]The core (underlined) is a combination of a trimeric coiled-coil
that contains a CTL epitope MEKLKELEK and the modified B-cell epitope
KLRNLEEELHSLRKNLNILNEELEELT (sequence 27 in Villard V. et al., PLoS ONE
2007, 2(7):e645), and the pentamer shown in Example 10 with excellent
panDR binding properties. At the C-terminal all six peptide chains have
the identical B-cell epitope, which is a tetra-repeat (NANP) of the
repetitive sequence of the circum sporozoite protein CS from Plasmodium
falciparum. At the N-terminus are about 18 different P. falciparum CTL
epitopes (U.S. Pat. Nos. 5,028,425, 5,972,351, 6,663,871) three different
epitopes per chain. The CTL-epitopes are separated by optimized
proteasomal cleavage sites (http://www.paproc2.de/paproc1/paproc1.html;
Hadeler K. P. et al., Math. Biosci. 2004, 188:63-79). Since the core of
these six peptide chains is identical, co-assembly of these six peptide
chains into one single SAPN allows the incorporation of about 18
different CTL epitopes into one single SAPN.

Example 14

Influenza Vaccine: HTL-Core, B-Cell Tetramer, CTL, Co-Assembly

[0167]The following is an example of an Influenza vaccine SAPN composed of
six peptide chains with an identical core and identical N-terminal B-cell
epitope M2e and about 20 CTL epitopes at the C-terminus (three or four
each per peptide chain) co-assembled into a single SAPN. The core
(underlined) is a combination of the trimer in Example 7 and the tetramer
in Example 9 with excellent panDR binding properties. The tetrameric
N-terminal B-cell epitope is the same as in Example 9. At the C-terminal
end the conserved CTL epitopes from Panda R. et al., Vaccine 2007,
25:7530-7539 (Table 15) are placed. Since the core of these six peptide
chains is identical, co-assembly of these six peptide chains into one
single SAPN allows the incorporation of about 20 different CTL epitopes
into one single SAPN.

[0168]The following is an example of an Influenza vaccine SAPN composed of
three peptide chains with an identical core and identical N-terminal
B-cell epitope M2e and nine B-cell epitopes at the C-terminus (three each
per peptide chain) co-assembled into a single SAPN.

[0169]The core (underlined) is a combination of the trimer in Example 8
and the tetramer in Example 9 with excellent panDR binding properties.
The tetrameric N-terminal B-cell epitope is the same as in Example 9. The
C-terminal B-cell epitopes are from Table 8 for H1, H2, H3, H5 consensus
1, H5 consensus 2, H7 consensus 1, H7 consensus 2, H7 consensus 3, H9
with negative charges between the epitopes to make the B-cell epitope
string less positively charged.